Production failures of bacterial cultures caused by bacteriophage infection are considered to be one of the major problems in industrial use of bacterial cultures. Bacteriophages have been found for many of the bacterial strains used in the industry, such as species of lactic acid bacteria e.g. Lactococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Streptococcus spp., Propionibacterium spp., Bifidobacterium spp, Staphylococcus spp. or Micrococcus spp. Furthermore, bacteriophage infections are also well known in other industrially useful species such as Bacillus spp., Enterobacteriaceae spp. including E. coli, Corynebacterium spp. Actinomycetes spp. and Brevibacterium spp.
In the food industry lactic acid bacterial starter cultures are widely used for food fermentations. It appears that among members of the lactic acid bacteria Lactococcus spp. are most devastated by bacteriophage infections. A factor, which leads to frequent bacteriophage infections in lactic acid bacterial starter cultures is the fact that the fermentation conditions in the food industry including the dairy industry are generally non-sterile. Thus, it has not yet been possible to eliminate bacteriophage contamination under these industrial conditions.
The lytic development of phages involves adsorption of the phages to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phages and release of progeny from the host. Cell-mediated mechanisms of interference with any of these events can prevent a phage infection. The ability of bacterial cultures to resist bacteriophage infection during industrial use depends to a large extent on host strain characteristics affecting one or more of the above mechanisms.
Thus, it has been shown that natural bacteriophage resistance or defense mechanisms exist in bacterial strains which ensure a certain level of protection against bacteriophage attack. These natural defense mechanisms include phage adsorption inhibition, prevention of phage DNA penetration, restriction of phage DNA and abortive infection.
The prevention of productive contact between phages and bacterial cells due to altered cell surface receptors for phages greatly reduces the ability of the phages to attack the cells. Adsorption of the phages to the cell surface is not always sufficient for the translocation of the phage DNA. It has been shown that host specific cell membrane proteins are involved in the prevention of phage DNA penetration.
Restriction/modification is a mechanism that operates by the cooperation of two enzyme systems, a DNA-cleaving restriction enzyme and a DNA-modifying enzyme, usually a methylase. The mechanism functions by cleaving the phage DNA, as it enters the cell.
Abortive phage infection is described as a mechanism that interferes with the phage development after phage expression has begun. This may eventually lead to a reduced level or termination of the production of viable phage progeny.
However, like many other traits of bacterial strains which are important for industrial performance, the above described natural phage defense mechanisms have been shown to be unstable characteristics, as they may be mediated by plasmids. Furthermore, these defense mechanisms are often phage specific, i.e. they are only active against a limited range of bacteriophage types. Accordingly, the prior art is not aware of a general and stably maintained host cell associated resistance mechanism against bacteriophage infection.
Based on the above natural defense mechanisms, the industry has designed and implemented strategies to possibly reduce bacteriophage infection of bacterial cultures including starter cultures for the fermentation of dairy products. Currently used strategies include the use of mixed starter cultures and alternate use of strains having different phage susceptibility profiles (strain rotation).
Traditionally, starter cultures in the dairy industry are mixtures of lactic acid bacterial strains. The complex composition of mixed starter cultures ensures that a certain level of resistance to phage attack is present. However, repeated subculturing of mixed strain cultures leads to unpredictable changes in the distribution of individual strains and eventually undesired strain dominance. This in turn may lead to increased susceptibility to phage attack and risk of fermentation failures.
Rotation of selected bacterial strains which are sensitive to different phages is another approach to limit phage development. However, it is difficult and cumbersome to identify and select a sufficient number of strains having different phage type profiles to provide an efficient and reliable rotation program. In addition, the continuous use of strains requires careful monitoring for new infectious phages and the need to quickly substitute a strain which is infected by the new bacteriophage by a phage resistant strain. In manufacturing plants where large quantities of bulk starter cultures are made ahead of time, such a quick response is usually not possible.
Studies have shown that a reduced growth capacity of a bacterial culture such as a proteinase-deficient lactic acid bacterium results in reduced phage proliferation (Richardson et al., 1983, 1984). However, such bacterial strains are still growing and are thus still susceptible to attack by phages.
Thus, the industry is not in the possession of any reliable strategy to secure that bacterial cultures used for industrial manufacturing of food products or other products are resistant against attack by bacteriophages. Furthermore, none of the currently used strategies prevent infections by any type of bacteriophages and none of these strategies are capable of precluding that bacteriophages, by a mutational event, circumvent the resistance mechanisms of the bacterial culture strains.
It is therefore a significant objective of the present invention to provide a method of preventing bacteriophage infection of bacterial cultures which are used in the manufacturing of food products and other products, wherein the cultures are completely resistant to attack by all types of bacteriophages.